Enzymatic reduction method for the preparation of compounds useful for preparing taxanes

ABSTRACT

An enzymatic reduction method, particularly a stereoselective enzymatic reduction method, for the preparation of compounds useful as intermediates in the preparation of taxanes.

This is a division of application Ser. No. 07/975,453, filed Nov. 12,1992 now U.S. Pat. No. 5,420,337.

FIELD OF THE INVENTION

The present invention relates to an enzymatic reduction method for thepreparation of compounds useful as intermediates in the preparation oftaxanes, and particularly to the stereoselective preparation of suchcompounds.

BACKGROUND OF THE INVENTION

Taxanes are diterpene compounds which find utility in the pharmaceuticalfield. For example, taxol, a taxane having the structure: ##STR1## wherePh is phenyl, Ac is acetyl and Bz is benzoyl, has been found to be aneffective anticancer agent.

Naturally occurring taxanes such as taxol may be found in plantmaterials, and have been isolated therefrom. Such taxanes may, however,be present in plant materials in relatively small amounts so that, inthe case of taxol, for example, large numbers of the slow-growing yewtrees forming a source for the compound may be required. The art hasthus continued to search for synthetic, including semi-synthetic routesfor the preparation of taxanes, such as taxol and analogs thereof, aswell as routes for the preparation of intermediates used in thepreparation of these compounds. Methods allowing efficient preparationof chiral intermediates, providing final taxane products having adesired stereoconfiguration, are particularly sought.

SUMMARY OF THE INVENTION

The present invention provides a method for the enzymatic reduction,preferably, the stereoselective enzymatic reduction, of ketogroup-containing compounds to form hydroxyl group-containingstereoisomers useful as intermediates in the preparation of taxanes suchas taxol.

Specifically, the present invention provides a method for the enzymaticreduction of a compound of the formula I or a salt thereof: ##STR2## toform a compound of the formula II or a salt thereof: ##STR3## where W is

(a) --NHR³ ; or

(b) --N₃ ;

R¹ is

(a) aryl;

(b) alkyl;

(c) alkenyl; or

(d) alkynyl;

R² is

(a) hydrogen; or

(b) R⁴ ;

R³ is

(a) hydrogen;

(b) R⁴ ;

(c) --C(O)--OR⁴ ; or

(d) --C(O)--R⁴ ; and

R⁴ is

(a) alkyl;

(b) aryl;

(c) cycloalkyl;

(d) alkenyl;

(e) alkynyl;

(f) cycloalkenyl; or

(g) heterocyclo;

where, with respect to the chiral center marked with an asterisk, saidcompound of the formula I may be present as a single isomer or as amixture of both R and S isomers (for example, as a racemate),

comprising the step of contacting said compound of the formula I or saltthereof with an enzyme or microorganism capable of catalyzing saidreduction, and effecting said reduction.

In a preferred embodiment of the present invention, the compound of theformula I or salt thereof is reduced to preferentially form thefollowing compounds IIa and/or IIb or salts thereof: ##STR4##

A particularly preferred embodiment of the present invention provides amethod for the stereoselective enzymatic reduction of a compound of theformula I or salt thereof to form a compound of the formula IIa or IIbor a salt thereof, comprising the step of contacting said compound ofthe formula I or salt thereof with an enzyme or microorganism capable ofcatalyzing said stereoselective reduction, and effecting said reduction.

DETAILED DESCRIPTION OF THE INVENTION

The methods of the present invention are described further as follows.

In the compounds of formula II, the group W and the hydroxyl group arebonded to asymmetric carbon atoms. Thus, the following fourstereoisomers may be formed as the compound of the formula II: ##STR5##

As used herein, "preferential" formation of the compounds of theformulae IIa and/or IIb denotes the formation of one or both of thesecompounds preferentially relative to formation of the compounds of theformulae IIc and/or IId.

The terms "stereoselective enzymatic reduction" and "stereoselectivereduction", as used herein, refer to the preferential formation of asingle enantiomer of the compound of the formula II (that is, IIa, IIb,IIc or IId) relative to other stereoisomers thereof. Thus, for example,stereoselective reduction of the compound of the formula I to form acompound of the formula IIa denotes prefential formation of the compoundof the formula IIa relative to the formation of compounds of theformulae IIb, IIc and IId. Stereoselective reduction of the compound ofthe formula I to form a compound of the formula IIb denotes preferentialformation of the compound of the formula IIb relative to compounds ofthe formulae IIa, IIc and IId.

Compounds of the formula IIa have the same absolute stereoconfiguration,at the carbon atom bearing the group W and the carbon atom bearing thehydroxyl group formed by the reduction process, as the compound(2R,3S)-(-)-N-benzoyl-3-phenylisoserine ethyl ester. Compounds of theformula IIb have the same absolute stereoconfiguration at thecorresponding carbon atoms as the compound(2S,3S)-(-)-N-benzoyl-3-phenylisoserine ethyl ester.

With respect to the chiral center marked with an asterisk, the startingcompound of formula I may be present as a single isomer having the R orS configuration, or as a mixture of the R and S isomers, for example, asa racemate.

The term "mixture", as said term is used herein in relation tostereoisomeric, such as enantiomeric compounds, includes mixtures havingequal (i.e. racemic for an enantiomeric mixture) or non-equal amounts ofstereoisomers.

The terms "enzymatic process" or "enzymatic method", as used herein,denote a process or method of the present invention employing an enzymeor microorganism.

The terms "alkyl" or "alk", as used herein alone or as part of anothergroup, denote optionally substituted, straight and branched chainsaturated hydrocarbon groups, preferably having 1 to 10 carbons in thenormal chain. Exemplary unsubstituted such groups include methyl, ethyl,propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl,heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl,undecyl, dodecyl and the like. Exemplary substituents may include one ormore of the following groups: halo, alkoxy, alkylthio, alkenyl, alkynyl,aryl, cycloalkyl, cycloalkenyl, hydroxy or protected hydroxy, carboxyl(--COOH), alkyloxycarbonyl, alkylcarbonyloxy, carbamoyl (NH₂ --CO--),amino (--NH₂), mono- or dialkylamino, or thiol (--SH).

The terms "lower alk" or "lower alkyl" as used herein, denote suchoptionally substituted groups as described above for alkyl having 1 to 4carbon atoms in the normal chain.

The terms "alkoxy" or "alkylthio" denote an alkyl group as describedabove bonded through an oxygen linkage (--O--) or a sulfur linkage(--S--), respectively. The term "alkyloxycarbonyl", as used herein,denotes an alkoxy group bonded through a carbonyl group. The term"alkylcarbonyloxy", as used herein, denotes an alkyl group bondedthrough a carbonyl group which is, in turn, bonded through an oxygenlinkage. The terms "monoalkylamino" or "dialkylamino" denote an aminogroup substituted by one or two alkyl groups as described above,respectively.

The term "alkenyl", as used herein alone or as part of another group,denotes such optionally substituted groups as described above for alkyl,further containing at least one carbon to carbon double bond.

The term "alkynyl", as used herein alone or as part of another group,denotes such optionally substituted groups as described above #or alkyl,further containing at least one carbon to carbon triple bond.

The term "cycloalkyl", as used herein alone or as part of another group,denotes optionally substituted, saturated cyclic hydrocarbon ringsystems, preferably containing 1 to 3 rings and 3 to 7 carbons per ring.Exemplary unsubstituted such groups include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl,cyclododecyl, and adamantyl. Exemplary substituents include one or morealkyl groups as described above, or one or more groups described aboveas alkyl substituents.

The term "cycloalkenyl", as used herein alone or as part of anothergroup, denotes such optionally substituted groups as described above forcycloalkyl, further containing at least one carbon to carbon double bondforming a partially unsaturated ring.

The terms "ar" or "aryl", as used herein alone or as part of anothergroup, denote optionally substituted, homocyclic aromatic groups,preferably containing 1 or 2 rings and 6 to 12 ring carbons. Exemplaryunsubstituted such groups include phenyl, biphenyl, and naphthyl.Exemplary substituents include one or more, preferably three or fewer,nitro groups, alkyl groups as described above or groups described aboveas alkyl substituents.

The terms "heterocyclo" or "heterocyclic", as used herein alone or aspart of another group, denote optionally substituted fully saturated orunsaturated, aromatic or non-aromatic cyclic groups having at least oneheteroatom in at least one ring, preferably monocyclic or bicyclicgroups having 5 or 6 atoms in each ring. The heterocyclo group may, forexample, have 1 or 2 oxygen atoms, 1 or 2 sulfur atoms, and/or 1 to 4nitrogen atoms in the ring. Each heterocyclo group may be bonded throughany carbon or heteroatom of the ring system. Exemplary heterocyclogroups include the following: thienyl, furyl, pyrrolyl, pyridyl,imidazolyl, pyrrolidinyl, piperidinyl, azepinyl, indolyl, isoindolyl,quinolinyl, isoquinolinyl, benzothiazolyl, benzoxazolyl, benzimidazolyl,benzoxadiazolyl, and benzofurazanyl. Exemplary substituents include oneor more alkyl groups as described above or one or more groups describedabove as alkyl substituents.

The terms "halogen" or "halo", as used herein alone or as part ofanother group, denote chlorine, bromine, fluorine, and iodine.

The term "taxane moiety", as used herein, denotes moieties containingthe core structure: ##STR6## which core structure may be substituted andwhich may contain ethylenic unsaturation in the ring system thereof.

The term "taxane", as used herein, denotes compounds containing a taxanemoiety as described above.

The term "hydroxy protecting group", as used herein, denotes any groupcapable of protecting a free hydroxyl group which, subsequent to thereaction for which it is employed, may be removed without disturbing theremainder of the molecule. Such groups, and the synthesis thereof, maybe found in "Protective Groups in Organic Synthesis" by T. W. Greene,John Wiley and Sons, 1981, or Fieser & Fieser. Exemplary hydroxylprotecting groups include methoxymethyl, 1-ethoxyethyl,1-methoxy-1-methylethyl, benzyloxymethyl,(β-trimethylsilylethoxy)methyl, tetrahydropyranyl,2,2,2-trichloroethoxycarbonyl, t-butyl(diphenyl)silyl, trialkylsilyl,trichloromethoxycarbonyl, and 2,2,2-trichloroethoxymenhyl.

The term "salt" includes acidic and/or basic salts formed with inorganicand/or organic acids and bases.

STARTING MATERIALS

The starting materials employed in the present reduction method may beobtained according to the following Reaction Scheme, and as described byCharles et al., J. C. S. Perkin I, 1139 (1980). ##STR7##

According to the above Reaction Scheme, compounds of the formula I maybe prepared by reacting a compound (i) with an oxalyl chloride ester ofthe formula Cl --C(O)--C(O)--OR², where R² is preferably unsubstitutedlower alkyl such as ethyl or methyl, for example, in anhydroustetrahydrofuran (THF) in the presence of 4-dimethylaminopyridine (DMAP)and pyridine, to form a compound (ii). compounds of the formula (i) andesters of the formula Cl --C(O)--C(O)--OR² are commercially available ormay readily be prepared by one of ordinary skill in the art. In thecompound (i), w is preferably an amide group --NH--C(O)--R⁴, such asbenzoylamino, or a urethane group --NH--C(O)--OR⁴, such as where R⁴ isunsubstituted alkyl (for example, the urethane groupt-butyloxycarbonylamino (BOC)), which may be prepared by reacting thecorresponding compound (i) where W is amino (--NH₂) with the reagent R⁴--C(O)--Cl or R⁴ --C(O)!₂ O.

A racemate of a compound of the formula I may then be prepared from thecompound (ii), for example, by heating the compound (ii) in ethanol inthe presence of anhydrous NaHCO₃ or other mild bases. Starting materialswhich are other than racemic may be obtained, for example, by separationof the isomers of the racemate prepared above, or by addition of one orboth of the enantiomers of the compound of formula I in unequal portionsto a racemic mixture thereof.

The present invention provides novel compounds of the formula (ii),where R¹, R² and W are as defined above, except that, when W is--NH--C(O)--R⁴ and R² is ethyl, (1) R⁴ is not isobutyl, n-propyl,cyclopentyl or phenyl when R¹ is methyl, and (2) R⁴ is not n-propyl whenR¹ is phenyl. Preferably, in the compounds of the formula (ii), R¹ isaryl such as phenyl, W is arylcarbonylamino such as benzoylamino oralkyloxycarbonylamino such as t-butyloxycarbonylamino (BOC), and R² isalkyl such as unsubstituted lower alkyl (e.g. methyl or ethyl). Allstereoisomers, such as cis- and transisomers, of the novel compounds ofthe formula (ii), alone or in admixture, are contemplated.

The present invention also provides novel compounds of the formula I,where R¹, R² and W are as defined above, except that, when W is--NH--C(O)--R⁴ and R² is ethyl, (1) R⁴ is not isobutyl, n-propyl,cyclopennyl or phenyl when R¹ is methyl, and (2) R⁴ is not n-propyl whenR¹ is phenyl. Preferably, in the compounds of the formula I, R¹ is arylsuch as phenyl, W is arylcarbonylamino such as benzoylamino oralkyloxycarbonylamino such as t-butyloxycarbonylamino, and R² is alkylsuch as unsubstituted lower alkyl (e.g. methyl or ethyl). Allstereoisomers of the novel compounds of the formula I, alone or inadmixture (e.g. racemates), are contemplated.

PREFERRED COMPOUNDS

It is preferred to prepare, according to the present invention,compounds of the formula II in which: W is --NHR³, R¹ is aryl,especially phenyl, R² is alkyl, especially unsubstituted lower alkylsuch as ethyl or methyl, and R³ is arylcarbonyl, especially benzoyl, oralkyloxycarbonyl, especially t-butyloxycarbonyl. It is further preferredto stereoselectively prepare compounds of the formula IIa or IIb.

ENZYMES AND MICROORGANISMS

The enzyme or microorganism employed in the present invention may be anyenzyme or microorganism capable of catalyzing the enzymatic reduction,preferably the stereoselective enzymatic reduction, described herein.The enyzmatic or microbial materials may be employed in the free stateor immobilized on a support such as by physical adsorption orentrapment.

Suitable enzymes, regardless of origin or purity, include those enzymesreferred to as oxido-reductases or dehydrogenases. The enzyme employedmay, for example, be an enzyme isolated from a microorganism such as byhomogenizing cell suspensions, followed by disintegration,centrifugation, DEAE-cellulose chromatography, ammonium sulfatefractionation, chromatography using gel filtration media such asSephacryl (cross-linked co-polymer of allyl dextran and N,N'-methylenebisacrylamide) chromatography, and ion exchange chromatography such asMono-Q (anion exchanger which binds negatively charged biomoleculesthrough quaternary amine groups) chromatography. Exemplary such enzymesinclude L-2-hydroxyisocaproate dehydrogenase, lactic acid dehydrogenase,yeast enzyme concentrate (may be obtained from Sigma), β-hydroxybutyratedehydrogenase, glucose dehydrogenase, alcohol dehydrogenase, glyceroldehydrogenase, formate dehydrogenase, pyruvate dehydrogenase, hydroxysteroid dehydrogenase, and those enzymes derived from the microorganismsdescribed following.

With respect to the use of microorganisms, the methods of the presentinvention may be carried out using any suitable microbial materialscapable of catalyzing the enzymatic reduction, preferably thestereoselective enzymatic reduction, described herein. For example, thecells may be used in the form of intact wet cells or dried cells such aslyophilized, spray-dried or heat-dried cells, or in the form of treatedcell material such as ruptured cells or cell extracts. Suitablemicroorganisms include genera from bacteria, yeasts and fungi such asAchromobacter, Acinetobacter, Actinomyces, Alkaligenes, Arthrobacter,Azotobacter, Bacillus, Brevibacterium, Corynebacterium, Flavobacterium,Methylomonas, Mycobacterium, Nocardia, Pseudomonas, Rhodococcus,Streptomyces, Xanthomonas, Aspergillus, Candida, Fusarium, Geotrichum,Hansenula, Kloeckera, Penicillium, Pichia, Rhizopus, Rhodotorula,Saccharomyces, Trichoderma, Mortierella, Cunninghamella, Torulopsis,Mucor and Rhodopseudomonas.

The use of genetically engineered organisms is also contemplated. Thehost cell may be any cell, e.g. Escherichia coli, modified to contain agene or genes for expressing one or more enzymes capable of catalysis asdescribed herein.

Preferred microorganisms include Arthrobacter simplex, Nocardiarestricta, Rhodococcus fascians, Mycobacterium vacca, Nocardiameditteranei, Nocardia autotrophica, Rhodococcus equi, Candida albicans,Pichia pastoris, Pichia metchanolica, Torulopsis polysporium, Torulopsisglabrata and Acinetobacter calcoaceticus, and especially Mortierellaalpina (e.g. ATCC 32221), Nocardia globerula (e.g. ATCC 21505),Cunninghamella echinulata (e.g. ATCC 26269), Nocardia salmonicolor (e.g.ATCC 19149), Geotrichum candidum (e.g. ATCC 34614), Candidaguilliermondii (e.g. ATCC 20318 and ATCC 9058), Aspergillus versicolor(e.g. ATCC 26268), Penicillium thomii (e.g. ATCC 14974), Rhodococcuserythropolis (e.g. ATCC 4277), Rhodococcus rhodochrous (e.g. ATCC 19150and ATCC 14341), Saccharomyces cerevisiae (e.g. ATCC 24702), Pseudomonasputida (e.g. ATCC 11172), Mortierella ramanniana (e.g. ATCC 38191),Mucor hiemalis (e.g. ATCC 8977B), Pichia pinus (e.g. ATCC 28780),Hansenula anomala (e.g. ATCC 8170), Hansenula fabiannii (e.g. ATCC58045) and Hansenula polymorpha (e.g. ATCC 26012).

Particularly preferred organisms for the preparation of compounds of theformula IIa are microorganisms of the species Hansenula polymorpha,especially the strain Hansenula polymorpha ATCC 26012, and the speciesHansenula fabiannii, especially the strain Hansenula fabiannii ATCC58045. It is also particularly preferred to employ cell extracts orisolated enzymes from these organisms.

The term "ATCC" as used herein refers to the accession number of theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, the depository for the organism referred

The enzymatic reduction method of the present invention may be carriedout subsequent to the fermentation of the microorganism employed(two-stage fermentation and reduction), or concurrently therewith, thatis, in the latter case, by in situ fermentation and reduction(single-stage fermentation and reduction). In the single-stage process,the microorganisms may be grown in an appropriate medium untilsufficient growth of the microorganisms is attained. A compound of theformula I may then be added to the microbial cultures and the enzymaticreduction continued with the fermentation, preferably until completeconversion is obtained.

In the two-stage process, the microorganisms may, in the first stage, begrown in an appropriate medium for fermentation until exhibiting thedesired enzymatic (e.g. oxido-reductase) activity. Subsequently, thecells may be harvested by centrifugation and microbial cell suspensionsprepared by suspending harvested cells in an appropriate bufferedsolution. Buffers such as tris-HCl, phosphates, sodium acetate and thelike may be used. Water may also be used to prepare suspensions ofmicrobial cells. In the second stage, the compound I may be mixed withthe microbial cell suspensions, and the enzymatic reduction of compoundI catalyzed by the microbial cell suspensions. The reduction ispreferably conducted until all or nearly all of the compound I isreduced.

Growth of the microorganisms may be achieved by one of ordinary skill inthe art by the use of an appropriate medium. Appropriate media forgrowing microorganisms include those which provide nutrients necessaryfor the growth of the microbial cells. A typical medium for growthincludes necessary carbon sources, nitrogen sources, and trace elements.Inducers may also be added. The term "inducer", as used herein, includesany compound enhancing formation of the desired enzymatic (e.g.oxido-reductase) activity within the microbial cell, such as thosecompounds containing keto groups. Formula I compounds may be added asinducers during growth of the microorganisms.

Carbon sources may include sugars such as maltose, lactose, glucose,fructose, glycerol, sorbitol, sucrose, starch, mannitol, propyleneglycol, and the like; organic acids such as sodium acetate, sodiumcitrate, and the like; amino acids such as sodium glutamate and thelike; and alcohols such as ethanol, propanol and the like.

Nitrogen sources may include N--Z amine A, corn steep liquor, soy beanmeal, beef extracts, yeast extracts, molasses, baker's yeast, trylptone,nutrisoy, peptone, yeastamin, sodium nitrate, ammonium sulfate and thelike.

Trace elements may include phosphates and magnesium, manganese, calcium,cobalt, nickel, iron, sodium and potassium salts.

The medium employed may include more than one carbon or nitrogen sourceor other nutrient.

Preferred media include aqueous media containing the following (inweight %):

    ______________________________________                                        Medium 1                                                                      Malt Extract           1%                                                     Yeast Extract          1%                                                     Peptone                1%                                                     Glucose                2%                                                                            pH 7.0                                                 Medium 2                                                                      Peptone                0.3%                                                   Glycerel               44                                                     Malt Extract           1%                                                     Yeast Extract          1%                                                                            pH 7.0                                                 Medium 3                                                                      Peptone                0.3%                                                   Fructose               2%                                                     Malt Extract           1%                                                     Yeast Extract          1%                                                                            pH 7.0                                                 Medium 4                                                                      Sodium Succinate       2%                                                     Malt Extract           1%                                                     Yeast Extract          1%                                                     Peptone                0.3%                                                                          pH 7.0                                                 ______________________________________                                    

The pH of the medium is preferably adjusted to about 6 to 8, mostpreferably to 6.5, sterilized, e.g. at a temperature of 121° C. for 30minutes, and then adjusted to a pH of about 6.5 to 7.5, preferably 7.0,after sterilization. The pH of the medium is preferably maintainedbetween 4.0 and 9.0, most preferably between 6.0 and 8.0, during thegrowth of microorganisms and during the reduction process.

Temperature is a measure of the heat energy available for the reductionprocess, and should be maintained to ensure that there is sufficientenergy available for this process. A suitable temperature range is fromabout 15° C. to about 60° C. A preferred temperature range is from about25° C. to about 40° C.

The agitation and aeration of the reaction mixture affects the amount ofoxygen available during the reduction process, which may be conducted,for example, in shake-flask cultures or fermentor tanks during growth ofmicroorganisms in a single-stage or two-stage process. The agitationrange from 50 to 1000 RPM is preferable, with 50 to 500 RPM being mostpreferred. Aeration of about 0.1 to 10 volumes of air per volume ofmedia per minute (i.e., 0.1 to 10 v/vt) is preferred, with aeration ofabout 5 volumes of air per volume of media per minute (i.e., 5 v/vt)being most preferred.

Complete conversion of the compound I may take, for example, from about12 to 48 hours, such as 4 to 24 hours, measured from the time ofinitially treating the compound I with a microorganism or enzyme asdescribed herein.

The enzymatic reduction method of the present invention may be carriedout using a co-factor such as nicotinamide adenine dinucleotide (NADH),especially when an isolated enzyme is employed. NADH, for example, maythereafter be regenerated and reused. A further enzyme that regeneratesthe NADH in situ may be employed such as formate dehydrogenase. Suitablehydrogen donors include molecular hydrogen, a formate (e.g. an alkalimetal or ammonium formate), a hypophosphite or an electrochemicalreduction in the presence of a viologen, for example methyl viologen. Itis also possible to regenerate NADH without further enzymes using, forexample, ethanol or formate.

It is preferred to employ an aqueous liquid as the reaction medium,although an organic liquid, or a miscible or immiscible (biphasic)organic/aqueous liquid mixture may also be employed.

It is preferred to employ 0.1 to 25 weight % of the compound I startingmaterial based on the combined weight of compound I and reaction medium.The amount of enzyme or microorganism employed relative to the startingmaterial is selected to allow catalysis of the enzymatic reduction ofthe present invention.

It is preferred to employ parameters, such as enzymes andmicroorganisms, which provide a stereoselective reduction. Astereoselective reduction is advantageous in that an efficientconversion of substrate may be achieved, and in that the procedureswhich may be employed in the subsequent separation of the desiredenantiomer of the formula II from the remaining components of thereaction medium may be minimized. It is particularly preferred to employparameters which provide a reaction yield greater than about 80%, mostpreferably greater than about 90%, and an optical purity greater thanabout 90%, most preferably greater than about 99%, of a desiredenantiomer of the formula II. To obtain stereoselective reduction of thesubstrate compound I, it is desirable to employ the enzymes andmicroorganisms indicated above as preferred.

SEPARATION

The products of the reduction process of the present invention may beisolated and purified, for example, by methods such as extraction,distillation, crystallization, and column chromatography.

For example, a preferred method for separating the compound IIa from theremaining components of the reaction medium is by extraction. Anexemplary extraction technique, such as where(2R,3S)-(-)-N-benzoyl-3-phenylisoserine ethyl ester is prepared by wholecell suspensions, is that where the reaction medium, containing theaforementioned suspensions, is extracted with ethyl acetate, the organiclayer is washed with brine, and the solvent is then removed underreduced pressure to generate an oily liquid which is chromatographed onsilica to produce the desired product compound IIa.

Taxanes are diterpene compounds containing the taxate moiety: ##STR8##described above. Of particular interest are taxanes containing a taxanemoiety in which the 11,12-positions are bonded through an ethyleniclinkage, and in which the 13-position contains a sidechain, whichtaxanes are exemplified by taxol. Pharmacologically active taxanes suchas taxol may be used as antitumor agents to treat patients sufferingfrom cancers such as breast, ovarian, colon or lung cancers, melanomaand leukemia.

The compounds of the formula II obtained by the reduction method of thepresent invention are particularly useful as intermediates in formingthe aforementioned sidechain on the taxate moiety. The addition of sucha sidechain, in and of itself, may impart an increased or more desirablepharmacological activity to the taxate product, or may form a taxaneproduct which is more readily converted to a taxane having an increasedor more desirable pharmacological activity than the starting compound.

The compounds of the formula II prepared according to the reductionmethod of the present invention may optionally be modified prior to usein sidechain formation. For example, compounds containing an azide group(N₃) as the group W may be treated with a reducing agent to form anamine group, the latter which may be substituted to form the group--NHR³.

The compounds of the formula II obtained by the method of the presentinvention may, for example, be used in the preparation ofsidechain-bearing taxanes such as those described in European PatentPublication No. 400,971, U.S. Pat. Nos. 4,876,399, 4,857,653, 4,814,470,4,924,012, and 4,924,011, all incorporated herein by reference.

For example, taxanes bearing a hydroxyl group at C-13, such as thosedescribed in the aforementioned European Patent Publication No. 400,971,may be coupled with an optionally modified compound of the formula II inthe presence of a condensing agent, for example, a carbodiimide such asdicyclohexylcarbodiimide or a reactive carbonate such as di-2-pyridylcarbonate, as well as a tertiary amine activating agent, for example, adialkylaminopyridine such as 4-dimethylaminopyridine. An inert solventsuch as benzene, toluene, a xylene, ethylbenzene, isopropylbenzene orchlorobenzene, and a temperature of from about 60° C. to about 90° C.,may be employed.

Coupling may be conducted as describedby Ojima et al., J. org. Chem.,56, 1681 (1991), incorporated herein by reference. See also Denis etal., J. Am. Chem. Soc., 110, 5917 (1988), also incorporated herein byreference. Taxol is preferably ultimately prepared as thesidechain-bearing taxane.

Salts or solyates such as hydrates of reactants or products may beemployed or prepared as appropriate in any of the methods of the presentinvention.

The present invention is further described by the following exampleswhich are illustrative only, and are in no way intended to limit thescope of the instant claims.

EXAMPLE 1 Preparation of Startina Material and Enzymatic ReductionPreparation of 2-Keto-3-(N-benzoylamino)-3-phenylpropionic acid ethylester: Racemic starting material

(a) Benzoyl phenylglycine ##STR9##

To (DL)-phenylglycine (9 g, 60 mmole) in aqueous NaOH (1N, 180 ml) at 0°C. was added dropwise neat benzoyl chloride (PhCOCl) (7.73 ml, 66 mmole)over a period of 5 minutes. The resulting solution was stirred for anadditional 1 hour. The reaction solution was washed with ethyl acetate(EtOAc) (20 ml×2), then neutralized by 6N HCl and extracted with EtOAc(60 ml×2). The combined EtOAc layer was washed with brine (30 ml×2),dried over MgSO₄, filtered and concentrated to give a residue. Theresidue was crystallized from EtOAc/hexane to give 10.65 g of benzoylphenylglycine as a white solid (70% yield, first crops). (The titleproduct is also commercially available.)

(b) 3-Benzoylamino-3-phenyl-(ethyl, 2-oxalyl) propenoic acid, ethylester ##STR10##

To a stirred solution of benzoyl phenylglycine prepared in step (a)(6.12 g, 24 mmole), 4-dimethylaminopyridine (100 mg, 0.82 mmole), andpyridine (5.86 ml, 72 mmole) in anhydrous tetrahydrofuran (THF) (24 ml)was added ethyl oxalyl chloride (5.35 ml, 48 mmole) at a rate toinitiate gentle refluxing. (Refluxing at this point was not criticalwhen sufficient refluxing (˜3.5 h) as followed was employed). Themixture was then heated to maintain a gentle reflux for 3.5 hours. Thereaction was monitored by thin layer chromatography (TLC) using 30%EtOAc in hexane as eluent (R_(f) for the starting material was on thebase line and R_(f) for the products were 0.50 and 0.63 (E and Zisomers)). After cooling, the room temperature mixture was treated withwater (48 ml) and stirred vigorously at room temperature for 1/2 hour.The resulting organic layer was separated and the aqueous layer wasextracted with EtOAc (36 ml×2). The combined organic layer was washedwith brine (30 ml×1), dried over Na₂ SO₄, filtered, concentrated, andcrystallized from EtOAc/hexane to obtain 4.68 g of the enol ester titleproduct (˜63% yield, first crop--no attempt was made to get a secondcrop.)

(c) Racemic 2-Keto-3-(N-benzoylamino)-3-phenylpropionic acid ethyl ester##STR11##

To a suspension of the enol ester title product prepared in step (b)above (6.0 g, 14.6 mmole) in 20 ml ethanol (EtOH) was added anhydrousNaHCO₃ (0.8 g, 9.49 mmole). The reaction mixture was refluxed for 1/2hour. The reaction was monitored by TLC using 2% acetone in CH₂ Cl₂ aseluent (R_(f) for the starting materials were 0.50 and 0.75 (E & Zisomers) and R_(f) for the product was 0.41). NaHCO₃ was filtered (ifany) and the filtrate was concentrated to an oil. It was purified bycolumn chromatography ((CO₂ Et)₂ was removed by column chromatography)(2% acetone/CH₂ Cl₂) to give 5.6 g of the title product (˜100% yield).(Crystallization was used for purification in subsequent preparation ofthe title product.) When the compound was stored in the freezer, itsolidified.

m.p.: 80°-83° C.

TLC: R_(f) =0.43; Silica gel; 2% Acetone in CH₂ Cl₂ ;

UV and PMA Visualization.

Enzymanic Reduction: Use of Various Strains of Whole Cells

The substrate for the following enzymatic reduction process was racemic2-keto-3-(N-benzoylamino)-3-phenylpropionic acid ethyl ester ("CompoundA") having the structure set forth above. Of particular interest in thisexample was preparation of the compound having the following structure:##STR12## and the name (2R,3S)-(-)-N-benzoyl-3-phenylisoserine ethylester ("Compound B"). The microorganisms which were employed in thereduction process are listed in Table 1 following.

The microorganisms employed were maintained in a vial in liquidnitrogen. For routine development of inoculum, one vial was inoculatedinto 100 ml of Medium 1 (see above for the composition thereof) in a 500ml flask and incubated at 28° C. and 280 RPM on a shaker for 48 hours.After growth of the microorganism, 10 ml of culture was inoculated intoa 500 ml flask containing 100 ml of Medium 1 and incubated at 28° C. and250 RPM on a shaker.

Cells were harvested and suspended in 100 mM potassium phosphate bufferpH 6.0. 10 ml of 20% w/v cell-suspensions were prepared.Cell-suspensions were supplemented with 25 mg of substrate (Compound A)and 750 mg of glucose and the reductions ("biotransformations") wereconducted at 25° C., 150 RPM for 72 hours. One volume of sample wastaken and extracted with two volumes of ethyl acetate and the separatedorganic phase was filtered through a 0.2 μm LID/x filter and collected.

Samples were analyzed for substrate and product concentration by aHewlett Packard 1070 HPLC System. A Phenomenex Cyanopropyl Column(150×4.6 mm, 5μ) was used. The mobile phase consisted of 5% isopropanolin hexane. The flow rate was 0.5 ml/min at ambient temperature. Thedetection wavelength was 230 nm. The retention times for substrate, syndiastereomer (both enantiomers) of product and anti diastereomer (bothenantiomers) of product were 26.8 min., 20.4 min., and 22.2 min,respectively.

The separation of the two enantiomers of the syn and anti diastereomerswas achieved by HPLC using dual columns connected in a series. The firstcolumn was a Pirkler column (DNBPG, dinitrophenylglycine) (250×4.6 mm,5μ) and the second column was Chiralcel OB (250×4.6 mm, 5μ) (bothcolumns purchased from J. T. Baker, Inc., Phillipsburg, N.Y.). Themobile phase consisted of 25:2.5:2.5:70 ofisopropanol:n-butanol:methanol: hexane. The flow rate was 0.5 ml/min.and the detector wavelength was 230 nm. The retention times for the twoenantiomers of syn were 20.1 min. and 23.3 min., respectively. Theretention times for the two enantiomers of anti were 22 min. and 27.8min., respectively.

The results obtained by using various microorganisms grown on Medium 1and following the above procedure are shown in Table 1.

Batches were also further purified, subsequent to extraction, asexemplified by the following procedure:

The reduction product subsequent to extraction isolation (0.822 g) wasdissolved in hot acetonitrile (16.5 ml) and the solution was filteredhot through a "D" sintered glass funnel. The filtrate was allowed tostand an room temperature (crystals formed quickly) for 45 minutes, andwas then allowed to stand at 4° C. It was filtered and washed with coldacetonitrile. The crystals were air dried and weighed. ¹ H NMR indimethylsulfoxide demonstrated that the crystals were essentially thesyn material. (Crystal weight: 0.38 g; α!^(D) 20 (Cl, CHCl₃)=-21.7;α!^(D) 20 (Cl, CH₃ OH)=-36.5).

The mother liquor residue (0.414 g) was dissolved in 7 ml of hotacetonitrile, filtered hot through a "D" sintered glass funnel andallowed to stand at room temperature for 2 hours. It was then placed ina cold room (4° C.) and left overnight. The crystals were then filteredand washed with cold acetonitrile (3×0.5 ml) and air dried giving 0.072g as a second crop. ¹ H NMR was consistent with >98% syn.

Analysis indicated that the crystals obtained were approximately 100%optically pure Compound B.

                  TABLE 1                                                         ______________________________________                                                          Reaction Yield                                                                            Optical Purity                                                    (syn compounds)                                                                           (Compound B)                                    Microorganism     (%).sup.1/  (%).sup.2/                                      ______________________________________                                        Candida guilliermondii ATCC 20318                                                               31          95                                              Rhodococcus erythropolis                                                                        39          96                                              ATCC 4277                                                                     Saccharomyces cerevisiae                                                                        35          94                                              ATCC 24702                                                                    Hansenula polymorpha ATCC 26012                                                                 98          99.5                                            Pseudomonas putida ATCC 11172                                                                   32          94                                              Nocardia globerula ATCC 21505                                                                   36          92                                              Mortierella ramanniana                                                                          35          97                                              ATCC 38191                                                                    Hansenula fabianii ATCC 58045                                                                   90          96                                              ______________________________________                                         .sup.1/ Reaction yield calculated as:                                         ##STR13##                                                                     .sup.2/ Optical purity calculated as:                                         ##STR14##                                                                     .sup.3/ Compound C had the following structure:                               ##STR15##                                                                

EXAMPLE 2 Use of Whole Cells: Variation in Reaction Time

The substrate for this process was Compound A. Of particular interest inthis example was preparation of Compound B. Both Compounds A and B aredescribed in Example 1.

Cells of Hansenula polymorpha ATCC 26012 were grown in 100 ml of Medium1 combined in 500 ml flasks. Growth was carried out at 25° C. for 48hours at 280 rpm. 100 ml of cultures were inoculated into 15 L of Medium2 (see above for the composition thereof) combined in a fermentor.Growth in the fermentor was carried out at 25° C., 15 liters per minutes(LPM) aeration and 500 RPM agitation for 60 hours. Cells were harvestedfrom the fermentor and used for the reduction ("biotransformation") ofCompound A to Compound B.

Cells (200 grams) were suspended in 1 liter of 100 mM potassiumphosphate buffer, pH 6.0 and homogenous cell suspensions were prepared.2.5 grams of Compound A and 35 grams of glucose were added to the cellsuspensions and the biotransformation of Compound A to Compound B wascarried out at 22° C., 160 RPM for 24 hours. After 24 hours, anadditional 35 grams of glucose were added and the biotransformation wascontinued for 72 hours at 22° C., 160 RPM. Samples were prepared andproduct yield and optical purity were determined as described inExample 1. The results obtained are summarized in Table 2 following.

                  TABLE 2                                                         ______________________________________                                        Reaction Time                                                                             Yield of     Optical Purity                                       (Hours)     syn compounds (%)                                                                          of Compound B (%)                                    ______________________________________                                        24          32           --                                                   48          65           --                                                   72          90           99.5                                                 ______________________________________                                    

EXAMPLE 3 Use of Cell Extracts and Co-factor

The substrate for this process was Compound A as described above. Ofparticlar interest in this example was preparation of Compound B alsodescribed above.

Cells of Hansenula polymorpha ATCC 26012 were grown on Medium 1 andMedium 2 as described in Example 2.

Cells (150 grams) were suspended in 1.5 L of 0.2M potassium phosphatebuffer, pH 6.0. The homogenized cell suspensions were disintegrated at4° C. by a Microfluidizer at 13,000 psi pressure. The disintegrated cellsuspension was centrifuged at 12,000 RPM for 30 minutes. The clearsupernatant ("cell extract") was used for the biotransformation ofCompound A to Compound B.

One liter of cell extract was supplemented with 2.5 grams of substrate(Compound A), formate dehydrogenase (500 units), 0.7 mM NAD⁺(nicotinamide adenine dinucleotide), and 25 grams of sodium formate. Thereaction was carried out in a pH stat at pH 6.0, 150 RPM agitation, and22° C. Periodically, samples were taken and analyzed for the reactionyield and optical purity of Compound B as described in Example 1. Theresults obtained are those shown in Table 3 following.

                  TABLE 3                                                         ______________________________________                                        Reaction Time                                                                            Compound B   Yield  Optical Purity                                 (Hours)    g/L          (%)    (%)                                            ______________________________________                                        48         2.2          88     >99%                                           ______________________________________                                    

In the above procedure, the NADH cofactor used for the biotransformationof Compound A to Compound B was, concurrent with the biotransformation,formed and regenerated using formate dehydrogenase, NAD⁺, and formate asshown below. ##STR16##

After complete conversion of Compound A to Compound B, the reactionmixture was adjusted to pH 7.0 and extracted three times with equalvolumes of ethyl acetate. The organic phase was separated and washedtwice with 0.7M sodium bicarbonate. The separated organic layer wasdried over anhydrous sodium sulfate and ethyl acetate was removed underreduced pressure. The resulting oily residue was dried under vacuum atroom temperature to recover a pale white solid in 85% yield (isolated)and 99% optical purity.

EXAMPLE 4 Use of Purified Oxido-Reductase

The substrate for this process (Compound A) was described in Example 1.Of particular interest in this example was the preparation of Compound Balso described in Example 1.

Growth of Hansenula polymorpha ATCC 26012 was carried out on Medium 1 asdescribed in Example 1. Cell extracts of Hansenula polymorpha ATCC 6012were prepared as described in Example 3.

Cell extracts (700 ml) were loaded onto a DEAE-cellulose (DE-52) columnand eluted with buffer containing sodium chloride in a linear gradientfrom 0 to 0.5M. Fractions containing oxido-reductase activity werepooled and concentrated by ammonium sulfate precipitation (70%saturation). Precipitated material was collected by centrifugation,dissolved in buffer, and loaded onto a Sephacryl S-200 column. Fractionscontaining reductase activity were pooled after chromatography andloaded onto a Mono-Q column. Proteins bound on the Mono-Q column wereeluted with a buffer containing sodium chloride in a linear gradientfrom 0 to 0.5M. Fractions having oxido-reductase activity were pooledand analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis. Thepurified enzyme was homogeneous. Overall, 250 fold purification wasachieved.

The transformation of Compound A to Compound B was carried out by thepurified enzyme (Mono-Q fraction). The reaction mixture in 20 ml of 0.1Mpotassium phosphate buffer (pH 6.0) contained 20 units of purifiedoxido-reductase enzyme, 200 mg of substrate (Compound A), 100 units offormate dehydrogenase, 1 gram of formate, and 50 mg of NAD⁺. Thereaction was carried out in a pH star at pH 6.0, 100 RPM agitation and22° C. for 48 hours. Product (Compound B) and substrate (Compound A)concentrations were determined by the procedures described in Example 1.After 48 hours of reaction time, an 89% reaction yield and greater than99% optical purity of Compound B was obtained.

EXAMPLE 5 Enzymatic Reduction

The substrate for this process (Compound A) is described in Example 1.Of particular interest in this example was the preparation of CompoundB, also described in Example 1.

Commmercially available oxido-reductases (5-20 units) were suspended in10 ml of 50 mM potassium phosphate buffer at pH 7.0. To the suspensionNADH or NADPH (1.5 mg/ml) was added. The reaction was started byaddition of 2 mg/ml of Compound A. The reaction was carried out at 25°C. at 150 RPM agitation for 48 hours. After 48 hours, samples were takenand analyzed for the reaction yield of Compound B as described inExample 1. The enzymes which produced Compound B and the reaction yieldsobtained are listed in the following Table 4.

                  TABLE 4                                                         ______________________________________                                                            Reaction Yield                                            Enzyme              (syn compounds)                                           ______________________________________                                        L-2-hydroxyisocaproate dehydro-                                                                   20                                                        genase                                                                        Lactic acid dehydrogenase                                                                         10                                                        Yeast enzyme concentrate                                                                          42                                                        β-hydroxybutyrate dehydrogenase                                                              5                                                         ______________________________________                                    

EXAMPLE 6

The substrate (Compound A) and method of bioreduction employed for thisexample were the same as those of Example 1. The organisms used and theresults obtained are listed in the following Table 5.

                  TABLE 5                                                         ______________________________________                                        Organism             Syn %.sup.1/                                                                            Anti %.sup.2/                                  ______________________________________                                        Candida guilliermondii ATCC 9058                                                                   18        82                                             Candida guilliermondi ATCC 20318                                                                   30.8      69.2                                           Penicillium thomii ATCC 14974                                                                      10        90                                             Rhodococcus rhodochorus ATCC 19150                                                                 14.4      85.6                                           Rhodococcus rhodochorus ATCC 14341                                                                 5.8       94.2                                           Mortierella alpina ATCC 32221                                                                      11        89                                             Hansenula anomala ATCC 8170                                                                        18.9      81.1                                           Cunninghamella echinulata ATCC 26269                                                               5.6       94                                             Pseudomonas putida ATCC 11172                                                                      30.1      69.9                                           Nocardia salmonicolor ATCC 19149                                                                   11        89                                             Geotrichum candidum ATCC 34614                                                                     12.1      87.9                                           Norcardia globerula ATCC 21505                                                                     35.5      64.5                                           Pichia pinus ATCC 28780                                                                            31        72                                             Aspergillus versicolor ATCC 26268                                                                  3.2       96                                             Mucor hiemalis ATCC 8977B                                                                          8         92                                             ______________________________________                                         ##STR17##                                                                     that is,  % (B + C)!-                                                         ##STR18##                                                                     that is,  % (D + E)!-  3/ Compound D had the structure:                       ##STR19##                                                                      4/ Compound E had the structure:                                               ##STR20##                                                                 

EXAMPLE 7

The substrate (Compound A) and method of enzymatic reduction employedfor this example were those of Example 5. The commercially availableenzymes used for this example, and the results obtained, are listed inthe following Table 6.

                  TABLE 6                                                         ______________________________________                                        Enzyme              Anti %.sup.1/                                             ______________________________________                                        Glucose dehydrogenase                                                                             88                                                        Alcohol dehydrogenase                                                                             81                                                        Glycerol dehydrogenase                                                                            72                                                        Formate dehydrogenase                                                                             78                                                        β-hydroxybutyrate dehydrogenase                                                              90                                                        Pyruvate dehydrogenase                                                                            92                                                        Hydroxy steroid dehydrogenase                                                                     87                                                        ______________________________________                                         .sup.1/ As defined above in Example 6.                                   

What is claimed is:
 1. A method for the enzymatic reduction of acompound of the formula I or a salt thereof: ##STR21## to form acompound of the formula II or a salt thereof: ##STR22## where W is(a)--NHR³ ; or (b) --N₃ ; R¹ is aryl; R² is(a) hydrogen; or (b) R⁴, whereR⁴ is (i) alkyl; (ii) aryl; (iii) cycloalkyl; (iv) alkenyl; (v) alkynyl;or (vi) cycloalkenyl, wherein the ring portion of said cycloalkyl andcycloalkenyl groups consists of 1 to 3 rings and 3 to 7 carbons perring; and R³ is(a) --C(O)--OR⁴, where R⁴ is alkyl; or (b) --C (O)--R⁴,where R⁴ is aryl; where, with respect to the chiral center marked withan asterisk, said compound of the formula I or salt thereof is employedas a starting material as a single isomer or as a mixture of both R andS isomers, comprising the step of contacting said compound of theformula I or salt thereof with an enzyme or microorganism whichcatalyzes said reduction, and effecting said reduction, therebyobtaining said compound of the formula II or salt thereof,(1) whereinsaid enzyme or microorganism is a microorgsnism selected from the generaconsisting of Candida, Rhodococcus, Saccharomyces, Hansenula,Pseudomonas, Nocardia, Mortierella, Penicillium, Cunninghamella,Geotrichum, Pichia, Aspergillus, and Mucor, or is an enzyme obtainedtherefrom, or (2) wherein said enzyme is selected from the groupconsisting of L-2-hydroxy isocaproate dehydrogenase, lactic aciddehydrogenase, β-hydroxyhutyrate dehydrogenase, glucose dehydrogenase,alcohol dehydrogenase, glycerol dehydrogenase, formate dehydrogenase,pyruvate dehydrogenase and hydroxy steroid dehydrogenase.
 2. The methodof claim 1, wherein said compound of the formula I or salt thereof isreduced to preferentially form the following compounds IIa and/or IIb orsalts thereof: ##STR23##
 3. The method of claim 2, comprising the stepof contacting said compound of the formula I or salt thereof with saidenzyme or microorganism which catalyzes stereoselective reductionthereof to form a compound of the formula IIa or salt thereof, andeffecting said stereoselective reduction.
 4. The method of claim 2,comprising the step of contacting said compound of the formula I or saltthereof with said enzyme or microorganism which catalyzesstereoselective reduction thereof to form a compound of the formula IIbor salt thereof, and effecting said stereoselective reduction.
 5. Themethod of claim 1, wherein W is --NHR³.
 6. The method of claim 5,wherein W is arylcarbonylamino.
 7. The method of claim 6, wherein W isbenzoylamino.
 8. The method of claim 7, wherein R¹ is phenyl and R² isalkyl.
 9. The method of claim 5, wherein W is alkyloxycarbonylamino. 10.The method of claim 9, wherein W is t-butyloxycarbonylamino.
 11. Themethod of claim 10, wherein R¹ is phenyl and R² is alkyl.
 12. The methodof claim 1, wherein, with respect to the chiral center marked with anasterisk, both the R and S isomers of the compound of formula I areemployed as the starting material in said method.
 13. The method ofclaim 12, wherein a racemate of R and S enantiomers is employed as saidstarting material.
 14. The method of claim 1, wherein, with respect tothe chiral center marked with an asterisk, a single R or S stereoisomer,but not both, of the compound of the formula I is employed as thestarting material in said method.
 15. The method of claim 1, whereinsaid enzyme or microorganism is a microorganism selected from the groupconsisting of Nocardia globerula, Penicillium thomii, Rhodococcusrhodochrous, Mortierella alpina, Hansenula anomala, Cunninghamellaechinulata, Nocardia salmonicolor, Geotrichum candidum, Pichia pinus,Aspergillus versicolor, Mucor hiemalis, Candida guilliermondii,Rhodococcus erythropolis, Saccharomyces cerevisiae, Pseudomonas putidaand Mortierella ramanniana, or is an enzyme obtained therefrom.
 16. Themethod of claim 15, wherein said enzyme or microorganism is amicroorganism selected from the group consisting of Nocardia globerulaATCC 21505, Candida guilliermondii ATCC 20318 and ATCC 9058, Rhodococcuserythropolis ATCC 4277, Saccharomyces cerevisiae ATCC 24702, Pseudomonasputida ATCC 11172, Mortierella ramanniana ATCC 38191, Penicillium thomiiATCC 14974, Rhodococcus rhodochrous ATCC 19150 and ATCC 14341,Mortierella alpina ATCC 32221, Hansenula anomala ATCC 8170,Cunninghamella echinulata ATCC 26269, Nocardia salmonicolor ATCC 19149,Geotrichum candidum ATCC 34614, Pichia pinus ATCC 28780, Aspergillusversicolor ATCC 26268, and Mucor hiemalis ATCC 8977B, or is an enzymeobtained therefrom.
 17. The method of claim 1, wherein said enzyme ormicroorganism is a microorganism of the species Hansenula polymorpha orHansenula fabianii, or is an enzyme obtained therefrom.
 18. The methodof claim 17, wherein the microorganism Hansenula polymorpha ATCC 26012,or an enzyme obtained therefrom, or the microorganism Hansenula fabianiiATCC 58045, or an enzyme obtained therefrom, is used.
 19. The method ofclaim 1, wherein said enzyme (2) is L-2-hydroxy isocaproatedehydrogenase, lactic acid dehydrogenase, β-hydroxybutyratedehydrogenase, glucose dehydrogenase, alcohol dehydrogenase, glyceroldehydrogenase, formate dehydrogensse, pyruvate dehydrogenase or hydroxysteroid dehydrogenase.
 20. The method of claim 1 further comprising,subsequent to said reduction, a separation step wherein said compound ofthe formula II is separated from other components of the reactionmedium.
 21. The method of claim 20, wherein said separation step is anextraction, distillation. crystallization, or column chromatographystep.
 22. The method of claim 21, wherein said separation step is anextraction step.
 23. The method of claim 21, wherein said separationstep is a crystallization step.
 24. The method of claim 21, wherein saidseparation step is a column chromatography step.
 25. The method of claim1, wherein said compound of the formula I is racemic2-keto-3-(N-benzoylamino)-3-phenylpropionic acid ethyl ester, and saidcompound of the formula II is (2R,3S)-(-)-N-benzoyl-3-phenyl-isoserineethyl ester.